Molecular Characterization of Fasciola spp. from Some Parts of Iran

Background: Identification of liver flukes, Fasciola hepatica, and Fasciola gigantica by morphometric parameters is not always reliable due to the overlapping measurements. This study aimed to characterize the liver flukes of animals from different parts of Iran by the genetic markers, ITS1, and COXI. Methods: We collected flukes from infected livestock in six provinces of Iran from Sep to Nov 2016. The flukes were identified by amplification of a 680 bp sequence of ITS1 locus followed by a restriction fragment polymorphism (RFLP) assay. The genetic diversity among isolates was evaluated by amplification and sequencing of a 493 bp fragment of the COXI gene. Results: We obtained 38 specimens from Khuzestan, 22 from Tehran, 10 from Isfahan, 10 from Mazandaran, 4 from Kurdistan, and 3 from Ardabil provinces. PCR-RFLP analysis revealed two patterns, representing F. hepatica, and F. gigantica. Fifty specimens from cattle and sheep exhibited F. hepatica pattern and 37 from the cattle, sheep, buffalo, and goat that of F. gigantica. The phylogeny based on COXI revealed two distinct clades separating F. hepatica from F. gigantica. In our phylogeny, the Iranian F. gigantica isolates showed a distinct separation from the African flukes, while grouped with the East Asia specimens demonstrating a common ancestor. The F. hepatica isolates clustered with the flukes from different parts of the world, including East Asia, Europe, and South America. Conclusion: The present study revealed a substantial genetic difference between F. gigantica populations of Asia and Africa, while F. hepatica isolates from different parts of the world shared high similarities.

2.4 to 17 million or even higher numbers depending upon the hitherto unknown situations in many countries are estimated to be infected (3). In the animal husbandry industry, the economic losses associated with this disease are at around two billion US dollars (1). Two flukes, Fasciola hepatica, and Fasciola gigantica are responsible for fascioliasis in humans and animals (1,4,5) with a higher severity for the latter species due to its bigger size and the greater body mass (6). In Iran, fascioliasis is an endemic disease of herbivores with prevalence ranging from 1.18% to 50% in different geographical regions (7)(8)(9)(10). The infection is of the higher rates among animals in the south of the country, while most human cases occur along the Caspian Sea littoral in the north. During 1988-1998, two significant outbreaks struck Gilan Province, infecting ≈15000 people (11,12). The Caspian Sea littoral has remained a hot spot for the disease. In the west and northwest, in Kermanshah and Ardabil provinces, human fascioliasis appears sporadically with limited outbreaks in the former one (13)(14)(15). Moreover, in the areas with high rates of the infection among local livestock, e.g., Lorestan, and Kohgiloye and Boyer-Ahmad, serology detected anti-Fasciola antibodies in humans (16,17). The flukes, F. hepatica, and F. gigantica are commonly identified based on morphologic and morphometric parameters (11). However, intermediate forms, presumably hybridizations of the two species, are hardly distinguishable by this approach (2). Reports of intermediate forms are available from different Asian countries, including China (18), Korea (19), Japan (20), Vietnam (21), and Iran (11,22), as well as Egypt in Africa. Today, various molecular markers, e.g., ITS1, ITS2, 28S rRNA, COXI, and NADI, are available for molecular identification of Fasciola spp. (2,4,18). Due to the conserved and variable regions and high copy numbers, ribosomal DNA (rDNA) has proved as a discriminating tool for identification of Fasciola species (2), whereas mtDNA sequences with higher mutation rates, lack of recombination and maternal inheritance serve as biomarkers for phylogenetic studies and genetic variability (23). In this study, by using the molecular markers, ITS1, and COXI, we characterized the liver flukes of livestock from different regions of Iran.

Study area
The samples were collected from six provinces of Iran with different geological and weather features including Ardebil in the northwest, Tehran in the north center, Isfahan in the center, Mazandaran in the north, Kurdistan in the west, and Khuzestan in the southwest during Sep to Nov 2016 (Fig. 1). Table 1 shows the climatological features in different regions of the study area.

Sample collection
We obtained 87 Fasciola flukes from different infected livestock, including sheep (n=29), goat (n=11), cattle (n=27), and Buffalo (n=20) slaughtered in the six provinces. The flukes were transferred to the Laboratory of Helminthology, School of Public Health, Tehran University of Medical Sciences, extensively washed with PBS and preserved in 70% alcohol, and kept at room temperature until used.

DNA extraction
Genomic DNA was extracted from a portion of the apical zone of the flukes. The tissue was ground using a surgical blade, and DNA extraction was performed by a commercial DNA extraction kit (Bioneer Corporation, Daejeon, South Korea) according to the manufacturer's instructions. The extracted DNAs were stored at -20 °C until used.

ITS1-PCR and RFLP analysis
A 680 bp fragment of ITS1 locus was targeted by using the primers (Table 2) designed by others (19) and synthesized in a commercial company (Macrogen Corporation, Seoul, South Korea). The 25 µl reactions contained 1 µl of the template DNA, 10 µl of master mix (0.2 U Taq DNA polymerase, 2 mM MgCl2, 400 pM dNTPs and the buffer system (Ampliqon, Skovlunde, Denmark), 200 pM each of forward and reverse primer, and double-distilled water (DDW) to the final volume. The PCR amplification programmed for an initial denaturation of 10 min at 94 °C followed by 25 cycles of 94 °C for 90 sec, 58 °C for 90 sec, and 72 °C for 90 sec with a final extension of 10 min at 72 °C. Amounts of 3 µl from amplicons were run on 1.5% gels at 90 V for 90 min, stained with 2% DNA safe stain® (Pishgam Biotech Co., Tehran, Iran) and visualized under UV (Syngene, Cambridge, UK). In all amplifications, DNA from a previously identified F. hepatica fluke (24) and DDW were included as positive and negative controls, respectively.  (25). The reactions contained 5 µl of PCR product, 5 µl of the enzyme, 2 µl of the buffer, and DDW to a final volume of 22 µl. The mixture incubated overnight at 37 °C followed by electrophoresis on 2% agarose gels and staining with 2% DNA safe stain. The ITS1 types were identified according to the generated patterns reported in other studies (25).

COXI amplification and phylogenetic analysis
The genetic diversity among Fasciola species was evaluated by amplification of a 493 bp sequence of COXI (20)

Blast analysis
The generated sequences were manually corrected and compared with similar sequences of the F. hepatica and F. gigantica available in GenBank database by the Basic Local Alignment Search Tool (BLAST) program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_ TYPE=BlastSearch)

Phylogeny
The COXI sequences generated in this study were aligned with similar sequences belonging to various Fasciola spp. isolates from Iran and other countries, including those representing the intermediate form. The distance between the sequences was calculated, and a phylogenetic tree was constructed by using the Jukes-Cantor option of the neighbor-joining method in a pairwise deletion procedure using MEGA 7 software (26).
The robustness of the topologies was estimated through 1,000 bootstrap replications.

Geographical analysis
ArcGIS 10.2 GIS software was used to draw maps of Iran and show the weather conditions, altitude, and average temperature.

ITS1-PCR and RFLP analysis
In PCR amplification, all the specimens and positive controls yielded the expected ≈680 bp amplicon. In RFLP analysis, the digestion generated two patterns: one comprised three bands of approximately 60 bp, 170 bp and 370 bp representing F. gigantica, and the other three bands of 60 bp, 100 bp, and 370 bp indicating F. hepatica (25). Of the 87 specimens, 50 (57.4%) from the cattle and sheep revealed the F. hepatica pattern, and 37 (42.6%) from the cattle, sheep, buffalo, and goat showed that of F. gigantica. No intermediate pattern was detected by this approach (Table 1 and Fig. 2).

BLAST analysis
In BLAST analysis, the F. hepatica COXI sequences generated here showed 98%-99% identity (97%-  (Fig. 3). Different phylogenetic approaches demonstrated almost the same topology. revealed two distinct types with some overlapping specific measurements indicating the intermediate form (11 (Fig. 1). These data are in agreement with the previous works exhibiting F. gigantica as the dominant species in the south, southwest, and southwest of Iran and F. hepatica as the more widespread species in north and northwest of Iran (9,22,30). In areas with a temperature gradient resulting from various altitudes, the two species exhibit distinct distributions. In Gilan Province, northern Iran, F. hepatica commonly occurs in the highlands while F. gigantica is more prevalent in the herbivores of the lowlands (11). We obtained F. hepatica flukes from areas with a mean elevation of 1127±80 m above sea level. In Khuzestan Province, a subtropical region with 19±00 m elevation, F. gigantica, was the dominant species, and F. hepatica was detected only in one sheep (Table 1, Fig. 4).   (31). In our phylogeny, the Iranian F. gigantica isolates showed a distinct separation from the African fluke and grouped with the East Asia specimens demonstrating a common ancestor. Our F. hepatica isolates clustered with the isolates from different parts of the world, including East Asia, Europe, and South America (Fig. 3). In our phylogenetic tree, a fluke from Japan previously reported a heterozygote with F. gigantica mtDNA background (20) grouped with F. hepatica clade reflecting the genuine maternal inheritance of this specimen (Fig. 3). The PCR-PFLP has shown a reliable and precise method for the detection of Fasciola species (25). In this study, PCR-PFLP detected no overlapping pattern indicating the intermediate forms.
One flaw in our study was the lack of specimens from Gilan Province, where morphometry previously identified the intermediate form.
Further studies with specimens from areas where two species co-occur (11) might reveal with more precision the molecular identity of Fasciola species in Iran and the possible occurrence of the intermediate form. Our study revealed F. hepatica as the primary cause of animal fascioliasis in Ardebil, Tehran, Isfahan, Mazandaran, and Kurdistan provinces, and F. gigantica as the common species in Khuzestan Province.

Conclusion
The present study revealed a substantial genetic difference between F. gigantica populations of Asia and Africa, and high genetic similarities between F. hepatica isolates from different parts of the world.

Ethical considerations
Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.